The present invention relates generally to methods and apparatus for delivery of macromolecules into cells. More specifically, the present invention provides methods and apparatus for delivering substances, such as macromolecules, e. g. polynucleotide vaccines (DNA vaccine and/or RNA vaccine) and protein-based vaccines, into selected cells in epidermal tissue with reduced sensation (reduced pain).
The first DNA vaccination procedure in the prior art was called naked DNA vaccination because a liquid solution of DNA was injected into the muscle of mice with no additives to enhance transfection. This method does transfect a few cells and does induce an immune response to the expressed antigen in mice. However, in humans and primates, the method does not work well.
In the prior art, an improvement in DNA vaccine efficiency was obtained by the use of a biolistic method for DNA delivery. The biolistic method is done by coating metal microbeads with DNA and shooting the particles into skin after accelerating the particles to a chosen velocity. This method works much better than naked DNA. Part of the reason is that the DNA coated particles are injected into the skin to a depth that increases the chance of transfecting Langerhans cells. However, the biolistic method has some disadvantages. First, it causes some skin damage that may scar in some individuals. Second, in spite of the increased efficiency, more efficiency is needed. Third, the ballistic particle remains inside the patient after treatment. In this respect, it would be desirable if a method for delivering DNA to biological cells were provided which does not cause skin damage that results in scarring. Also, it would be desirable if a method for delivering DNA to biological cells were provided which does not leave a residue of ballistic particles in cells that are treated. As a matter of interest, the following U.S. patents disclose biolistic methods: U.S. Pat. Nos. 5,036,006 and 5,478,744.
A number of additional approaches to delivering macromolecules to biological cells are disclosed in the prior art and are represented by the following U.S. patents or other publications as follows.
U.S. Pat. No. 5,019,034 of Weaver et al discloses a process for electroporation of tissues in which electrodes are placed on top of the tissue surface, such as skin, of a patient. Molecules that are used for treating the skin are placed in reservoirs on top of the skin surface, and the treatment molecules must penetrate into the skin tissues transdermally. That is, the treatment molecules must pass from outside the skin to inside the skin. Not only is the surface layer of the skin relatively impermeable, if the layers of the skin to be treated are near the basal lamina of the epidermis, then the treatment molecules must traverse considerable skin tissue before the cells to be treated are reached by the treatment molecules. Such a treatment method is inefficient for treating cells near the basal lamina. Rather than using electrodes that are placed on the skin surface and have treatment molecules pass through the skin transdermally to treat biological cells near the basal lamina of the epidermis, it would be desirable if an electroporation method were provided for delivering molecules to biological cells in the epidermis, near the basal lamina, without having the treatment molecules pass through the skin transdermally.
U.S. Pat. No. 5,273,525 of Hofmann discloses an apparatus for electroporation of drugs and genetic material into tissues which employs a hollow hypodermic needle for placing the drugs and genetic material in the vicinity of the tissues to be electroporated. Whenever a hollow hypodermic is employed in a tissue, the tissue is cut with a circular cut by the hollow hypodermic needle. As a result, when a patient receives hypodermic injection, the patient has considerable pain. To avoid such a circular cut, and to avoid the considerable pain involved, it would be desirable if a method for delivering molecules to biological cells were provided which does not employ a hypodermic needle.
U.S. Pat. No. 5,318,514 of Hofmann discloses an applicator for the electroporation of drugs and genes into cells. The applicator includes a plurality of needle electrodes which can be penetrated into the skin of a patient. Material to be electroporated into the skin is retained in a fluid reservoir which wets an open cell foam elastomer carrier for the fluid. Because the material to be electroporated is retained in a fluid, in both the reservoir and the open cell foam elastomer, careful control of the amount of the material at the electrode surfaces is difficult. It is difficult to control how much fluid flows down from the reservoir and the open cell foam elastomer to the surfaces of the needle electrodes, and, thereby, it is difficult to control how much of the treatment molecules is actually present on the surfaces of the electrodes 16 as the electroporation process is being carried out on the patient. Moreover, the presence of the fluid medium can have a flushing or washing effect on the tissues that are electroporated in such a way that the electroporation process is interfered with. In these respects, it would be desirable if an electroporation method for delivering molecules to biological cells were provided which does not employ a fluid medium that flows down onto the electrodes as the electroporation process is being carried out on the patient.
Other disclosures relating to the use of electroporation to mediate gene transfer into epidermal cells are found in an article by Reiss et al entitled xe2x80x9cDNA-mediated gene transfer into epidermal cells using electroporationxe2x80x9d in Biochem. Biophys. Res. Commun., Vol. 137, No. 1, (1986), pages 244-249 and in an article by Titomirov entitled xe2x80x9cIn vivo electroporation and stable transformation of skin cells of newborn mice by plasmid DNAxe2x80x9d in Biochim. Biophys. Acta., Vol. 1088, No. 1, (1991), pages 131-134.
U.S. Pat. No. 5,389,069 of Weaver discloses a method and apparatus for in vivo electroporation of tissues which employs a hollow cylindrical needle for providing treating substances deep into tissues. As mentioned above, avoiding the use hollow cylindrical needles would be desirable to avoid the pain involved therewith.
U.S. Pat. Nos. 5,580,859 and 5,589,466, both of Felgner et al, disclose a method of delivering macromolecules into muscles and skin of a patient by an injection method. Their method does not employ electroporation.
U.S. Pat. No. 5,697,901 of Eriksson discloses gene delivery into tissues by the use of a gene-carrying fluid medium that is pressurized in conjunction with hollow microneedles. Problems of control and flushing using fluid media have been discussed hereinabove. An electroporation step is not employed in the Eriksson patent. As a matter of interest, Eriksson addresses the subject of pain in two respects. There is a statement that the hollow microneedle system can be used for treating pain. There is a statement that pain in wounds can be relieved by cooling. It is noted by the present inventors herein that Eriksson does not discuss his treatment method per se as being of a pain free or reduced pain treatment method. The present inventors theorize that the pressurized fluid injection method that is employed by Eriksson is not conducive to a pain free or reduced pain treatment method. In this respect, it would be desirable to provide a gene therapy treatment method that employs micro-sized needles, but that does not employ a pressurized fluid injection step for injecting fluid into a patient.
In an article by Henry et al entitled xe2x80x9cMicrofabricated Microneedles: A Novel Approach to Transdermal Drug Deliveryxe2x80x9d in Journal of Pharmaceutical Sciences, Vol. 87, No. 8, August 1998, pages 922-925, there is a disclosure that an array of microneedles are employed to penetrate the epidermis to leave micro-sized perforations to facilitate transdermal permeability of fluid-carried treatment agents into the microperforated epidermis. Because the microneedles are inserted only a microscopic distance into the epidermis, use of the microneedles is potentially nonpainful. There is no disclosure that the microneedles are to be used as electrodes. Also, an electroporation step is not disclosed in the Henry et al article.
Further with respect to the issue of reduced pain treatment, it is noted that two important electrical parameters in electroporation are closely related to a perceived pain in vivo. One parameter is absolute voltage experienced by the in vivo tissue. Another parameter is the pulse width experienced by the in vivo tissue. In these respects, it would be desirable to provide an electroporation method for delivering molecules to biological cells which applies relatively low absolute voltage to cells undergoing electroporation and which can be used, if desired, to apply pulses having relatively short pulse width to the cells undergoing electroporation.
Still other features would be desirable in a method and apparatus for delivery of macromolecules into epidermal cells. For example, when electrodes are penetrated into the epidermis, the conductive base electrode portions and the conductive tips of the electrodes may exhibit electrical characteristics which are undesirable with respect to the electroporation process in general and the biological cells that are treated in particular. In this respect, it would be desirable if a method and apparatus for delivery of macromolecules into epidermal cells were provided which render nonconductive the base portions and tip portions of the electrodes.
Once electrode assemblies having a plurality of needle electrodes have been employed on a patient, it may be a difficult task to clean and sterilize them for a subsequent use. In this respect, it would be desirable if a method and apparatus for delivery of macromolecules into cells were provided in which the electrode assemblies are disposable.
When disposable electrode assemblies are employed, it would be desirable if the disposable electrode assemblies are packaged in sterile packaging.
Thus, while the foregoing body of prior art indicates it to be well known to use electroporation to deliver molecules to biological cells, the prior art described above does not teach or suggest a method and apparatus for delivery of macromolecules into cells which has most of the following combination of desirable features: (1) does not cause skin damage that results in scarring; (2) does not leave a residue of ballistic particles in cells that are treated; (3) provides an electroporation method for delivering molecules to biological cells in the epidermis, near the basal lamina, without having the treatment molecules pass through the skin transdermally; (4) does not employ a hypodermic needle; (5) does not employ a fluid medium that flows down onto the electrodes as the electroporation process is being carried out on the patient; (6) does not employ a pressurized fluid injection step for injecting fluid into a patient; (7) applies relatively low absolute voltage to cells undergoing electroporation; (8) if desired, can be used to apply pulses having relatively short pulse width to the cells undergoing electroporation; (9) renders the base portions and tip portions of the electrodes nonconductive; (10) provides disposable electrode assemblies; and (11) provides electrode assemblies which are packaged in sterile packaging. The foregoing desired characteristics are provided by the unique method and apparatus for delivery of macromolecules into cells of the present invention as will be made apparent from the following description thereof. Other advantages of the present invention over the prior art also will be rendered evident.
In accordance with one aspect of the invention, a method is provided for delivery of molecules into biological cells which includes the steps of:
(a) coating electrodes in an electrode assembly with the molecules to be delivered,
(b) attaching the electrode assembly having the coated electrodes to an electrode assembly holder,
(c) providing a waveform generator,
(d) establishing electrically conductive pathways between the electrodes and the waveform generator,
(e) locating the electrodes such that the biological cells are situated therebetween, and
(f) providing pulse waveforms from the waveform generator to the electrodes, such that molecules on the electrodes are driven off of the electrodes and delivered into the biological cells.
The pulse waveforms may be provided by applying a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, to the biological cells. The sequence of at least three DC electrical pulses has one, two, or three of the following characteristics (a) at least two of the at least three pulses differ from each other in pulse amplitude, (b) at least two of the at least three pulses differ from each other in pulse width, and (c) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses.
Additionally, the method can include a step of providing the electrode assembly holder with electrically conductive pathways between the electrode assembly and the waveform generator.
In addition, the method can include a step of providing the electrode assembly in a sterile package. In such a case, the electrode assembly is removed from the sterile package prior to use.
Further, the method can include the steps of providing the electrodes with electrically insulated outer surface electrode tip portions and electrically insulated outer surface electrode base portions.
The molecules in the electrode coating can be in a solid phase. The molecules in the electrode coating are, preferably, macromolecules. The macromolecules in the electrode coating can include a polynucleotide vaccine (DNA vaccine and/or RNA vaccine) or a protein-based vaccine.
With a variation of the method of the invention, the molecules can be delivered to Langerhans cells in epidermal tissue of a patient with reduced sensation (reduced pain or nearly painless or pain free) to the patient. To provide reduced sensation delivery of molecules to the patient, the following conditions are maintained (a) the pulse waveforms have an absolute applied voltage in a range of 0.1 to 300 volts; (b) the electrodes of opposite polarity are separated by a separation distance in a range of from 50 to 500 microns; and (c) the electrodes are penetrated into the epidermal tissue a distance up to and slightly beyond the basal lamina layer of the epidermal tissue.
The pulse waveforms which drive the coating molecules off of the electrodes are electrophoresis waveforms. The pulse waveforms which deliver the driven-off molecules into the biological cells are electroporation waveforms. Generally, common pulse waveforms both drive the coating molecules off of the electrodes and deliver the driven-off molecules into the biological cells.
The biological cells can be in vivo, ex vivo, or in vitro. More specifically, the biological cells can be in epidermal tissue and can be Langerhans cells in the epidermal tissue.
In accordance with another aspect of the invention, an apparatus is provided for delivery of molecules into biological cells and includes a waveform generator which provides pulse waveforms. An electrode assembly holder is provided, and an electrode assembly is mechanically supported by the electrode assembly holder. The electrode assembly holder is also electrically connected to the waveform generator through electrically conductive pathways. The electrode assembly includes electrodes which are coated with the molecules to be delivered into the biological cells.
The electrode assembly can be removable and replaceable from the electrode assembly holder. In this respect, the electrode assembly includes electrode-assembly-conductive strips. The electrode assembly holder includes holder conductors which are registrable with the electrode-assembly-conductive strips when the electrode assembly is mechanically connected to the electrode assembly holder. Also, the electrode assembly holder includes electrically conductive pathways between the holder conductors and the waveform generator.
With the apparatus, a sterile package can be provided for the electrode assembly. The sterile package is removed from the electrode assembly after the electrode assembly is mechanically supported by the electrode assembly holder and is electrically connected to the waveform generator.
With the apparatus, if desired, the waveform generator provides pulse waveforms which include a sequence of at least three single, operator-controlled, independently programmed, DC electrical pulses, to the biological cells. The sequence of at least three DC electrical pulses has one, two, or three of the following characteristics (a) at least two of the at least three pulses differ from each other in pulse amplitude, (b) at least two of the at least three pulses differ from each other in pulse width, and (c) a first pulse interval for a first set of two of the at least three pulses is different from a second pulse interval for a second set of two of the at least three pulses.
The electrodes can include electrically insulated outer surface electrode tip portions and electrically insulated outer surface electrode base portions. The electrodes are coated with macromolecules, which may include a polynucleotide vaccine (a DNA vaccine and/or a RNA vaccine) and/or a protein-based vaccine. The polynucleotide vaccine or protein-based vaccine can be a solid form, coating the electrodes, prior to using the electrodes on a patient.
In accordance with yet another aspect of the invention, a packaged sterile electrode assembly is provided which includes a sterile electrode assembly which includes electrodes which are coated with the molecules to be delivered into biological cells. The electrode assembly includes electrode-assembly-conductive strips for connection to complementary electrically conductive pathways leading to the waveform generator. In addition, an internally sterile package encloses the sterile electrode assembly contained therein.
With the packaged sterile electrode assembly, the electrodes can include electrically insulated outer surface electrode tip portions and electrically insulated outer surface electrode base portions.
With the packaged sterile electrode assembly, the electrodes are coated with macromolecules which can include a solid phase polynucleotide (DNA vaccine and/or RNA vaccine) and/or a solid phase protein-based vaccine.
In accordance with the invention, transfection of cells with DNA in vivo, using electric field mediated transfection, is an efficient process. Additionally, electric fields can be used for the delivery of other macromolecules such as RNA and proteins into cells. In the prior art, the electric field delivery has one disadvantage, that being the pain induced by the high voltage electrical pulses required for the transfection. In contrast, as described herein, a method is provided for delivering macromolecules (DNA, RNA, and protein) to cells, in tissues in vivo, using painless (or nearly painless) and efficient electric field mediated delivery.
The electric fields required for electric field mediated DNA transfection, by electroporation, are in the range of 100 volts per cm to 20,000 volts per cm. In accordance with the invention, using a maximum inter-electrode gap of 500 microns (0.5 mm), this range is converted to a range of absolute voltages in a range of 5 to 1,000 volts. Taking into account electrophoresis voltage for driving the DNA off of the electrodes, the range of absolute voltages is in a range of 0.1 to 1,000 volts.
Pain sensation resulting from an electrical stimulus is dependent upon several factors. Among these are voltage, amperage, pulse rise time, pulse width, and pulse frequency. Above a threshold value applied directly to tissues in vivo, increased voltage or increased pulse width will result in an increase in pain.
Two threshold values are important in painless in vivo electric field mediated delivery of macromolecules. A first threshold is the threshold for pain. It is desirable to reduce absolute voltage (and, if desired, also pulse width) applied directly to tissues in vivo below the threshold value for pain. However an opposing second threshold is the minimal electric field required for electric field mediated delivery of macromolecules. Usually, increasing pulse parameters beyond this second threshold value results in increased efficiency in the delivery of macromolecules. An excessive increase will result in cell death.
Fortunately, the important electric field parameter for electric field mediated delivery of macromolecules is volts per cm rather than absolute voltage. To maintain a constant electric field, as the distance between electrodes decreases, the absolute voltage needs to be decreased. Similarly, as the distance between electrode decreases, if the absolute voltage stays the same, the electric field increases. Thus narrowing the distance between electrodes permits a decrease in absolute voltage applied directly to tissues in vivo.
In addition to absolute voltage, pulse width is also important. Very narrow pulse widths significantly increase the threshold voltage required for pain sensation. Electroporation efficiency, within certain limits, is proportional to the product of pulse voltage multiplied by the pulse width. This means that if voltage can be increased, the pulse width can be decreased. Therefore, a decrease in inter-electrode distance also will allow decreased pulse widths to be used.
Another advantage of reduced inter-electrode distance is that fewer dermal nerves are located between the electrodes for the portions of the electrodes that may penetrate through the epidermis into the dermis. For a given applied voltage, the fewer nerves between the electrodes, the less pain is perceived.
Another factor affecting pain is the proximity of the electric field to nerve endings. In the skin, nerves and nerve endings exist throughout the dermis but are absent in the epidermis. However, in the upper papillary layer of the dermis, nerves are relatively few and far between. This means that electrodes that only penetrate the epidermis or epidermis plus upper dermis (papillary layer of dermis) will not be near many nerves. Thus, very short electrodes would induce almost no pain upon insertion nor would pain be induced upon the application of an electric field since the majority of the field is between the electrodes, which are located in the epidermis.
A number of applications of the method and apparatus for delivery of macromolecules into cells, of the invention, are contemplated. Briefly, such applications include polynucleotide vaccination, protein vaccination, and gene therapy.
For DNA vaccination, there are two overriding requirements. One is gene expression in vivo and the other is that at least some of the cells expressing the antigen need to be antigen-presenting cells. The highest concentration of accessible antigen presenting cells resides in the skin as cells called Langerhans cells. These cells are part of a very effective group of antigen presenting cells called dendritic cells. Electroporation is a viable alternative method for transfecting selected cells in vivo.
Proteins also can be introduced into cells using electric field mediated delivery. In conventional vaccination, proteins are delivered outside cells using a hypodermic needle. This type of delivery is inefficient in inducing a cell mediated cytotoxic lymphocyte immune response. Some infectious diseases require a cytotoxic lymphocyte response as a component of the immune response for efficient clearance of the infection. Delivery of proteins into cells promotes the induction of that response.
Delivery of therapeutic genetic medicine into cells for the purpose of making those cells express a missing protein is the basis of gene therapy. The method and apparatus of the present invention can be used to deliver therapeutic DNA into cells on the surface of any accessible organ in addition to the skin. The method of the invention is a method for painless, effective delivery of macromolecules to tissues, in vivo, for the purpose of vaccination (or treatment), DNA vaccination, gene therapy, or other reasons. An electrode with at least one of two characteristics is used for delivery of macromolecules into cells in tissue. One of the two characteristics is an electrode length short enough that it does not penetrate to a depth in tissue with nerve endings. Another characteristic is that inter-electrode distances are small enough to allow pulse parameters (voltage and pulse width) to be used that are painless. Only one or the other of these characteristics is needed in any given application, however, they may be used together.
The above brief description sets forth rather broadly the more important features of the present invention in order that the detailed description thereof that follows may be better understood, and in order that the present contributions to the art may be better appreciated. There are, of course, additional features of the invention that will be described hereinafter and which will be for the subject matter of the claims appended hereto.
In this respect, before explaining a preferred embodiment of the invention in detail, it is understood that the invention is not limited in its application to the details of the construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood, that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting.
As such, those skilled in the art will appreciate that the conception, upon which disclosure is based, may readily be utilized as a basis for designing other structures, methods, and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention.
In view of the above, it is an object of the present invention to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not cause skin damage that results in scarring.
Another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not leave a residue of ballistic particles in cells that are treated.
Even another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that provides an electroporation method for delivering molecules to biological cells in the epidermis, near the basal lamina, without having the treatment molecules pass through the skin transdermally.
Still a further object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not employ a hypodermic needle.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that does not employ a fluid medium that flows down onto the electrodes as the electroporation process is being carried out on the patient.
Still another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which does not employ a pressurized fluid injection step for injecting fluid into a patient.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that applies relatively low absolute voltage to cells undergoing electroporation.
Still a further object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that can be used, if desired, to apply pulses having relatively short pulse width to the cells undergoing electroporation.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which renders the base portions and tip portions of the electrodes nonconductive.
Still a further object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells that provides disposable electrode assemblies.
Yet another object of the present invention is to provide a new and improved method and apparatus for delivery of macromolecules into cells which electrode assemblies are packaged in sterile packaging.
These together with still other objects of the invention, along with the various features of novelty which characterize the invention, are pointed out with particularity in the claims annexed to and forming a part of this disclosure. For a better understanding of the invention, its operating advantages and the specific objects attained by its uses, reference should be had to the accompanying drawings and descriptive matter in which there are illustrated preferred embodiments of the invention.